Sodium/proton antiporter gene

ABSTRACT

The present inventors successfully cloned the rice Na + /H +  antiporter gene. It is possible to produce salt tolerant plants by using the isolated gene, or genes with equivalent functions.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of PCT/JP99/07224 filed Dec.22, 1999; which claims priority to Japanese Application No. 10/365604,filed Dec. 22, 1998.

TECHNICAL FIELD

The present invention relates to a novel Na⁺/H⁺ antiporter derived fromplants and the DNA encoding the antiporter, as well as methods forproducing and using the same.

BACKGROUND ART

Salt tolerance of plants is important to both agriculture andenvironmental protection. Today, one third of the land on earth is saidto be dry land. Further, it is anticipated that the proportion of dryland will increase in the future, due to the progressive desertificationof both cultivated land and green land. Considering the prediction thatthe world population in the year 2050 will be 1.5 times that of todayand the serious problems of provisions arising as a result, developmentof cultivars that grow on land ill-fitted for cultivation, especially ondry land, as well as cultivation techniques for the same is a matter ofgreat urgency. The problem with agriculture on dry land is saltaccumulation. In a dry climate, evapotranspiration outstripsprecipitation and continued irrigation on land where much is desired fordrainage leads to plenty of salt accumulation, due to the deposition ofsalt on the surface by acceleration of rise in subterranean water levelthat bear salinity. Examples where cultivation becomes impossible as aresult are known from the ancient past, represented by the end ofTigris-Euphrates civilization. The problem still arises today. Thus,innovation of agriculture, on dry land and on land where salt isaccumulated, to enhance the salt tolerance of plants is of greatimportance (Toshiaki Tanno (1983) Kagaku to Seibutsu 21:439-445 “Salttolerance of crops and mechanism of the same”; Yasutaka Uchiyama (1988)Kagaku to Seibutsu 26:650-659 “Agricultural use of salinenvironment”).

There are two kinds of stress related to salt stress against plants,namely stress by osmotic pressure and stress by ionicity. An osmoticpressure stress is a stress whose action is the same as the stress bydehydration. It results from high osmotic pressure, due to high salinityenvironment around the plant, which leads to a setback of waterabsorption of the plant and at the same time deprivation of water fromthe plant body. It is known that a mechanism exists in the plant toavoid the osmotic pressure stress. The core substances associated withthis function are ions (such as K⁺, Na⁺, Cl⁻, organic acid, etc.) aswell as substances called compatible solutes. The term “compatiblesolute” refers to substances such as sugar, proline (a kind of aminoacid), and glycine betaine (a quaternary ammonium compound), and so on,which do not disturb the metabolic pathway or inhibit enzymatic action,even when accumulated at a high concentration in the cell. Plant cellsaccumulate these substances which, in turn, preserve the osmoticpressure balance to the external world (Manabu Ishitani, Keita Arakawa,and Tetsuko Takabe (1990) Chemical Regulation of Plants 25:149-162,“Molecular mechanism of salt tolerance in plants”).

Almost no development has been made regarding the mechanism of plants toavoid ionic stress. Absorption of excess Na⁺ by the plant cell leads toinhibition of intracellular enzyme reaction and finally to metabolictrouble (Toru Matoh (1997) Chemical Regulation of Plants 32:198-206,“Salt tolerance mechanism of the plant”). Therefore, it is necessary toeliminate the intracellulary accumulated Na⁺ from the cell or isolate itinto intracellular organs, such as vacuoles. The Na⁺/H⁺ antiporter(sodium/proton antiporter) is assumed to play the central role in thisprocess. The Na⁺/H⁺ antiporters of plant cells are thought to exist onboth the cell membrane and the vacuolar membrane. They utilize the pHgradient formed between the biomembranes by the H⁺ pump (H⁺-ATPase andH⁺-PPase), an element that transports H⁺ as the energy to transport Na⁺existing in the cytoplasm out of the cell or into the vacuole. Moreover,it is presumed that plants contacted with salt of high density, have toretain intercellular K⁺/Na⁺ ratio high enough, maintaining the osmoticpressure balance between the cell exterior and interior by accumulatingNa⁺ in the vacuole through the Na⁺/H⁺ antiporter.

The Na⁺/H⁺ antiporters found existing on plasma membrane are wellexamined in animals, yeasts, bacteria and so on. On the plasma membraneof an animal cell, H⁺ is carried by the Na⁺/H⁺ antiporter, to maintainthe balance of H⁺ in the cell, utilizing the Na⁺ concentration gradientbetween the membranes formed by Na⁺/K⁺-ATPase. Therefore, the antiporteris presumed to be deeply related with intracellular pH modulation,control of the cell volume, as well as Na⁺ transport through the plasmamembrane (Orlowski, J. and Grinstein, S. (1997) J.Biol.Chem.272:22373-22376; Aronson, P. S. (1985) Ann.Rev.Physiol. 47:545-560).Na⁺/H⁺ antiporters exist in various cells of animals and six isoforms(NHE 1 to 6) have been reported (Orlowski, J. and Grinstein, S. (1997)J.Biol.Chem 272:22373-22376).

The first gene cloned for yeast was the gene (sod2) from fission yeast(Schizosaccharomyces pombe), which was cloned as a gene related to Na⁺transport and salt tolerance (Jia, Z. P., McCullough, N., Martel, R.,Hemmingsen, S. and Young, P. G. (1992) EMBO J. 11:1631-1640). Also, agene with high identity to this gene has been found from a budding yeast(Saccharomyces cerevisiae), as well as Zygosaccharomyces rouxii (namedNHA1 and ZSOD2, respectively) (Prior, C. et al. (1996) FEBS Letter387:89-93; Watanabe, Y. et al. (1995) Yeast 11:829-838). Two differentNa⁺/H⁺ antiporter genes (nhaA, nhaB) have been isolated from E. coli(Karpel, R. et al. (1988) J.Biol.Chem. 263:10408-10410; Pinner, E. etal. (1994) J.Biol.Chem. 269:26274-26279), each closely related to Na⁺transport and salt tolerance. With respect to plants, activities inalgae and such have been examined (Katz, A. et al. (1989)Biochim.Biophys.Acta 983:9-14).

On the other hand, there are only reports on activity in plants forantiporters restricted on vacuolar membranes. To date, Na⁺/H⁺antiporters on the vacuoles have been investigated in connection withsalt tolerance in halophytes growing in an environment with highsalinity (Matoh, T. et al. (1989) Plant Physiol. 89:180-183; Hassidim,M. et al. (1990) Plant Physiol. 94:1795-1801; Barkla, B. J. et al.(1995) Plant Physiol. 109:549-556), as well as in glycophytes with highsalt tolerance, like barley and sugar beet (Hassidim, M. et al. (1990)94:1795-1801; Blumwald, E. et al. (1987) Plant Physiol. 85:30-33;Garbarino, J. and DuPont, F. M. (1988) Plant Physiol. 86:231-236;Garbarino, J. and DuPont, F. M. (1989) Plant Physiol. 89:1-4; Staal, M.et al. (1991) Physiol.Plant. 82:179-184). The above findings indicatethat Na⁺/H⁺ antiporters are closely related to salt tolerance of plants.There are several reports on characteristics of Na⁺/H⁺ antiporters onthe vacuolar membrane. The Km of the antiporter activity for Na⁺ isabout 10 mM similar to that on cytomembrane of mammals (Blumwald, E. etal. (1987) Plant Physiol. 85:30-33; Garbarino, J. and DuPont, F. M.(1988) Plant Physiol. 86:231-236; Orlowski, J. (1993) J.Biol.Chem.268:16369-16377). Moreover, it is known that amiloride and amiloridederivatives, which are specific inhibitors of Na⁺ transporters, inhibitthe Na⁺/H⁺ antiporters on the plant vacuolar membrane and that on themammalian plasma membrane in a competitive manner (Blumwald, E. et al.(1987) Plant Physiol. 85:30-33; Orlowski, J. (1993) J.Biol.Chem.268:16369-16377; Tse, C. M. et al. (1993) J.Biol.Chem. 268:11917-11924;Fukuda, A. et al. (1998) Plant Cell Physiol. 39:196-201). These findingssuggest the characteristic similarities between Na⁺/H⁺ antiporter on thevacuolar membrane of plants and that on mammalian plasma membrane. Thereare various reports on Na⁺/H⁺ antiporter activity of plants as mentionedabove, however, in spite of the various trials done, analysis of thesubstantial part, namely genes as well as proteins thereof, were stillleft behind (Katz, A. et al. (1989) Biochim.Biophys.Acta 983:9-14;Barkla, B. and Blumwald, E. (1991) Proc.Natl.Acad.Sci.USA88:11177-11181; Katz, A., Kleyman, T. R., and Pick, U. (1994)Biochemistry 33:2389-2393).

Recently, a gene expected to encode a protein that shares amino acidsequence homology with known Na⁺/H⁺ antiporter has been cloned fromArabidopsis; however, the function of this gene remains to be resolved(M. P. Apse et al. (1998) Final Programme and Book of Abstracts “11thInternational Workshop on Plant Membrane Biology”, Springer; C. P.Darley et al. (1998) Final Programme and Book of Abstracts “11thInternational Workshop on Plant Membrane Biology”, Springer).

Examples of Na⁺/H⁺ antiporter genes isolated from plants are only thoseisolated from Arabidopsis, a dicotyledon, described above. No isolationof genes from monocotyledoneae, including species such as rice andmaize, which are industrially useful crops, have been reported untilnow.

DISCLOSURE OF THE INVENTION

Of all the important crops, rice is a crop with low salt tolerance. Itsgrowth is inhibited to the halves with 150 mM NaCl as compared tobarley, which is a highly salt tolerant crop, and shows inhibition ofthe same level with 250 mM NaCl. Garbarino et al. reported thesuppression of Na⁺ flow to the shoot by accumulating Na⁺ in the vacuoleof the root might increase salt tolerance of barleys (Garbarino, J. andDuPont, F. M. (1988) Plant Physiol. 86:231-236). From verifying thisfact, it has been known that the Na⁺/H⁺ antiporter activity of thebarley root vacuolar membrane increases through treatment with salt. Ithas also been known that barley has far and away a higher activity thanrice (Garbarino, J. and DuPont, F. M. (1988) Plant Physiol. 86:231-236;Fukuda, A. Yazaki, Y., Ishikawa, T., Koike, S., and Tanaka, Y. (1998)Plant Cell Physiol. 39:196-201).

On the contrary, the activity does not rise in rice even if it istreated with salt (Fukuda, A. Yazaki, Y., Ishikawa, T., Koike, S., andTanaka, Y. (1998) Plant Cell Physiol. 39:196-201). Further, Na⁺transport from root to the shoot of rice is known to be higher than thatof the phragmites, which belong to the Gramineae family, like rice, andshows higher salt tolerance (Matsushita, N. and Matoh, T. (1991)Physiol.Plant. 83:170-176). Therefore, it is possible that the strengthof Na⁺/H⁺ antiporter activity of the root vacuolar membrane is deeplyassociated with rice salt tolerance. These reports indicate that itmight be possible to increase salt tolerance of rice by rising Na⁺/H⁺antiporter activity in the rice root. On this account, there was adesire to isolate genes that might increase Na⁺/H⁺ antiporter activityof rice.

This situation led to the present invention, an object of which is toprovide an Na⁺/H⁺ antiporter derived from monocotyledoneae, preferablyrice, and gene(s) encoding the same, as well as a method for producingand using the same. The present invention provides use of the gene forproduction of salt tolerant plants as a favorable use of the presentDNA.

The present inventors identified a cDNA clone from rice anthotaxy thatshares homology with the Na⁺/H⁺ antiporter (NHX1) gene from the buddingyeast by analyzing a base sequence from the GeneBank higher plantsdatabase. Using this sequence as a probe, the present inventorssucceeded in newly cloning the full-length gene designated “OsNHX1”,which is expected to encode the Na⁺/H⁺ antiporter of rice.

The isolated OsNHX1 cDNA is approximately 2.3 kb and is presumed toencode a protein of 535 amino acids (FIG. 1). From an amino acidhydrophobicity analysis, the protein was detected to have 12transmembrane regions (FIG. 2).

The amino acid sequence predicted from OsNHX1 was detected to havesignificant identity with the amino acid sequence of NHX1 and mammalianNa⁺/H⁺ antiporter (NHE)(Table 1). Specifically, high identity was seenin the transmembrane region supposed to be involved in ion transport(FIG. 3).

These three proteins (NHX1 from budding yeast, NHE6 from mammals, andOsNHX1) turned out to form a cluster, according to the dendrogram formedfor various Na⁺/H⁺ antiporters reported to date (FIG. 4). The OsNHX1protein of the present invention is expected to be expressed inintracellular organs, such as vacuoles, and play an important role inthe Na⁺ transport of the vacuolar membrane, due to the report that NHX1protein is expressed in the late endosome (Nass, R. and Rao, R. (1998)J.Biol.Chem. 273:21054-21060) and the indication that NHE6 protein isalso expressed in the cell (Numata, M., Petrecca, K., Lake, N. andOrlowski J. (1998) J.Biol.Chem. 273:6951-6959).

Further, the present inventors succeeded in obtaining transgenic plantsby transferring the isolated OsNHX1 gene into the rice callus andredifferentiating them utilizing Agrobacterium method.

The present invention relates to a novel Na⁺/H⁺ antiporter derived frommonocotyledoneae and the DNA coding said antiporter, as well as methodsfor production and use, especially for the production of salt tolerantplants using same. More specifically, the present invention provides thefollowing:

-   -   (1) a DNA selected from the group consisting of:        -   (a) a DNA encoding the protein consisting of the amino acid            sequence described in SEQ ID NO: 2, and        -   (b) a DNA comprising the coding region of the base sequence            described in SEQ ID NO: 1;    -   (2) a DNA encoding the Na⁺/H⁺ antiporter derived from        monocotyledoneae selected from the group consisting of:        -   (a) a DNA encoding the protein consisting of the amino acid            sequence described in SEQ ID NO:2, wherein one or more amino            acids are substituted, deleted, inserted and/or added, and        -   (b) a DNA hybridizing under a stringent conditions to the            DNA consisting of the base sequence described in SEQ ID            NO:1;    -   (3) the DNA of (2), wherein the monocotyledoneae is a plant        belonging to the Gramineae family;    -   (4) a vector comprising the DNA of (1) or (2);    -   (5) a transformant cell having the DNA of (1) or (2), or the        vector of (4);    -   (6) the transformant cell of (5), wherein the cell is a plant        cell;    -   (7) a protein encoded by the DNA of (1) or (2);    -   (8) a method for production of the protein of (7), which        comprises the steps of:        -   cultivating the transformant cell of (5), and recovering the            expressed protein from said cell or the supernatant of the            culture thereof;    -   (9) a transformant plant comprising the transformant cell of        (6);    -   (10) the transformant plant of (9), wherein the plant is a        monocotyledon;    -   (11) the transformant plant of (10), wherein the plant is a        plant belonging to the Gramineae family;    -   (12) the transformant plant of (11), wherein the plant is rice;    -   (13) a transformant plant that is the offspring or clone of the        transformant plant of any of (9) to (12);    -   (14) a material for the breeding of the transformant plant of        any of (9) to (13);    -   (15) an antibody that binds to the protein of (7);    -   (16) a nucleic acid molecule that hybridizes with the DNA        described in SEQ ID NO: 1, and which has a chain length of at        least 15 nucleotides.

The present invention provides a novel Na⁺/H⁺ antiporter derived frommonocotyledoneae, as well as a DNA encoding the same. The base sequenceof the cDNA encoding the Na⁺/H⁺ antiporter “OsNHX1”, derived from riceand isolated by the present inventors, is indicated in SEQ ID NO: 1. Theamino acid sequence of the protein encoded by the cDNA is described inSEQ ID NO: 2.

The “OsNHX1” gene showed significant identity with many known amino acidsequences of the Na⁺/H⁺ antiporters, and especially high identity wasobserved at sites related to ion transport. This finding suggests that“OsNHX1” protein plays an important role in Na⁺ transport in rice. It issupposed that Na⁺/H⁺ antiporters of plants are involved in thesecurement of osmotic pressure balance in the plant body under a highsalinity stress. Thus, it is anticipated that the “OSNHX” geneespecially can be applied to production of salt tolerant cultivars.

Not only “OsNHX1” protein, but also proteins with equivalent functions,are included in this invention. The term “proteins with equivalentfunctions to ‘OsNHX1’ protein” herein means that the object proteinfunctions as an Na⁺/H⁺ antiporter. The activity of an Na⁺/H⁺ antiportercanbe detected, for example, by detecting the H⁺ ejection from thebiomembrane vesicle due to addition of Na⁺ as the recovery offluorescence, by monitoring H⁺ concentration gradient between isolatedbiomembrane vesicle formed by H⁺-ATPase as the fluorescence extinctionof acridine orange (Fukuda, A., Yazaki, Y., Ishikawa, T. Koike, S., andTanaka, Y. (1998) Plant Cell Physiol. 39:196-201).

In one embodiment, the protein with equivalent function to “OsNHX1” is amutant protein having amino acid sequence with one or more amino acidsubstitution, deletion, insertion and/or addition to the amino acidsequence of “OsNHX1” protein (SEQ ID NO: 2), and which retainsequivalent functions with “OsNHX1” protein. Such proteins can beprepared, for example, according to the following method. A methodinducing mutations in the amino acid of “OsNHX1” can be mentioned as onemethod well known to ordinary skilled in the art. That is, one ordinaryskilled in the art can prepare a modified protein with equivalentfunctions to “OsNHX1” by modifying the amino acid sequence of “OsNHX1”protein (SEQ ID NO: 2). For example, by utilizing a site-directedmutagenesis method (Kramer, W. & Fritz, H. -J. “Oligonucleotide-directedconstruction of mutagenesis via gapped duplex DNA” Methods in Enzymology154:350-367, 1987) and such with the purpose to increase proteinactivity or the like. Mutations of amino acids also happen to occur innature. The protein of this invention include proteins having amino acidsequence with 1 or more amino acids substitution, deletion, insertion oraddition to the natural amino acid sequence of “OsNHX1” protein (SEQ IDNO: 2), and that retain equivalent functions to natural proteins,regardless whether they are artificial or derived from nature. There isno limitation on the part or the number of amino acid in the protein tobe modified, so long as the modified protein retains equivalentfunctions with the natural “OsNHX1” protein. Generally, aminoacidmodifications are done to amino acids within 100 amino acids,preferably within 50 amino acids, much more preferably within 20 aminoacids, and most preferably within 5 amino acids.

In another embodiment, the protein having equivalent functions with“OsNHX1” protein is a protein encoded by a DNA derived frommonocotyledoneae that hybridizes to the DNA encoding “OsNHX1” protein(SEQ ID NO: 1), having an equivalent function with “OsNHX1” protein.Techniques such as hybridization techniques (Southern, E. M.: Journal ofMolecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction(PCR) techniques (Saiki, R. K. et al. Science, Vol.230, 1350-1354, 1985;Saiki, R. K. et al. Science, Vol.239, 487-491, 1988) can be mentioned astechniques known to those skilled in the art for preparing proteins.That is, it is routine for a person skilled in the art to isolate a DNAwith high identity to the “OsNHX1” gene from rice or other monocotyledonand obtain proteins with an equivalent function to “OsNHX1” protein fromthat DNA, using the base sequence of “OsNHX1” gene (SEQ ID NO: 1) orparts thereof as a probe, and oligonucleotides hybridizing specificallyto the base sequence of “OsNHX1” gene (SEQ ID NO: 1) as a primer. Suchproteins, derived from monocotyledoneae with an equivalent function tothe “OsNHX1” protein, obtainable by hybridization technique or PCRtechnique, are included in the proteins of this invention.

Monocotyledoneae, preferably plants belonging to the Gramineae familycan be mentioned as plants used as the source of genes for isolation byhybridization techniques and PCR techniques. For example, besides rice,barley (Hordeum vulgare), wheat (Triticum aestivum), maize (Zea mays)and so on can be mentioned, as plants belonging to the Gramineae family.However, it is not limited to them.

Methods for isolating DNA encoding proteins with an equivalent functionto the “OsNHX1” protein using the above-described techniques include,for example, but are not limited to, the following. For example,hybridization of cDNA or genomic libraries, prepared frommonocotyledoneae with probes (for example, DNA consisting of the basesequence described in SEQ ID NO: 1 or parts thereof) labeled with ³²Pand such, is carried out. Conditions for hybridization using ³²P labeledprobes are 25° C. (without formamide) as a mild condition and usually42° C., employing hybridization solutions (50% formamide, 5×SSPE,2×Denhard's solution, 0.1% (w/v) SDS, and 100 μg/ml of herring sperm DNA(Sambrook J, Fritsch E F, Maniatis T (1989) Molecular cloning: ALaboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.),2^(nd) Ed.)). Prehybridization is carried out at a minimum for more thanan hour, and hybridization is performed for 24 hours. Washing of thehybridized filter is carried out at 25° C. (wash solution: 2×SSC, 0.1%SDS) for a mild condition (a condition with low stringency), at 42° C.(wash solution: 2×SSC, 0.1% SDS) for an ordinary condition, and at 56°C. (wash solution: 0.1×SSC, 0.1% SDS) for a stringent condition (acondition with high stringency).

If the protein encoded by the DNA isolated as above has an equivalentfunction as “OsNHX1” protein, it generally shows a high amino acidsequence identity with “OsNHX1” protein. The term “high identity” asused herein refers to an identity higher than at least 60%, preferablyhigher than 80%, more preferably higher than 85%, and much morepreferably higher than 90%. The amino acid sequence identity iscalculated, for example, by a homologyanalysis program (Lipman, D. J.and Pearson, W. R. (1985) Science 227, 1435-1441) supplied by GENETYXsoftware (Software development corporation).

The protein of the present invention can be prepared by methods known tothose skilled in the art as recombinant proteins or as natural proteins.Recombinant proteins can be prepared, for example, by inserting a DNAencoding the protein of the present invention into an adequateexpression vector, transfecting an appropriate cell with the vector andpurifying the protein from the transformant cell, as described later on.Alternatively, natural proteins can be prepared, for example, byexposing cell extracts, prepared from cells that express the protein ofthe present invention (for example, rice cells), to affinity columns towhich antibodies, prepared by immunizing appropriate immune animals withprepared recombinant proteins or partial peptides thereof, are attached,and purifying the proteins bound to the column.

Additionally, the present invention provides DNAs encoding the proteinsof the present invention described above. The DNAs of the presentinvention includes genomic DNAs, cDNAs, and chemosynthetic DNAs and soon, and can be any DNA without limitation, so long as it encodes aprotein of the present invention. The base sequence of the “OsNHX1”cDNA, included in the present invention, is shown in SEQ ID NO: 1.

The genomic DNA, as well as the cDNA can be prepared according toconventional methods, known to those skilled in the art. Genomic DNA,for example, can be isolated using PCR, by designing appropriate primersfrom the base sequence information of the gene of the present invention,and then screening a genomic library using the obtained amplified DNAfragment as a probe. Alternatively, for example, it is possible toisolate the cDNA from a cDNA library according to the same manner.

The DNA of the present invention can be, for example, utilized inpreparation of recombinant proteins, as well as in production oftransformant plants with salt tolerance. In preparing recombinantproteins, generally, a DNA encoding the protein of the present inventionis inserted into an appropriate expression vector, the expression vectoris transferred into an appropriate cell, the transformed cell iscultivated and the expressed protein is purified.

Recombinant proteins can be prepared, for example, by transferringvectors, having DNAs encoding the protein of the present inventioninserted therein, into cells, such as bacterial cells, like E.coli,yeast cells, insect cells, mammalian cells, and so on, by known genetransfer methods, like the electroporation method, the calcium phosphatetransfection method and such, then expressing the recombinant proteinsin the cell. Recombinant proteins expressed in the host cell can bepurified according to methods known to those skilled in the art. Forexample, it is possible to express the protein as a fusion protein, withglutathione S-transferase (GST), using vectors such as pGEX (Pharmacia)in E. coli, and purify it using a glutathione column (Shigeo Ohno andYoshifumi Nishimura (1997) “Cell Engineering supplement: Protocol ofprotein experiments” Shujun-sha).

Moreover, to prepare transformant plants using the DNA of the presentinvention, a DNA encoding the protein of the present invention isinserted into an appropriate vector, the vector is transferred into aplant cell, and the obtained transformed plant cell is regenerated. Thetransfer of the plant expression vector into the plant cell can be donefor example, according to the species, through methods utilizingAgrobacteriums or methods involving the direct transfer into the cell.Methods that utilize Agrobacteriums, for example, are methods of Nagelet al. (Microbiol. Lett. 67:325(1990)) and methods of Raineri et al. forrice (Bio/Technology 8:33-38(1990)). These are methods in whichAgrobacteriums are transformed with plant expression vectors (pUC systemand so on. For example, pCAMBIA vector (Medical Research Council),etc.), and the transformed Agrobacteriums are transferred to plant cellsusing standard methods, like the leefdisk method, the callus method andso on. Methods for the directly transferring a plant expression vectorinto a cell include the electroporation method, the particle gun method,the calcium phosphate method, the polyethylene glycol method and so.

There is no limitation on the plant cells to which vectors of theinvention may be transferred, but monocotyledonous, preferably plantsbelonging to the Gramineae family are mentioned. Plants, like maizeexcept rice, can be mentioned as plants belonging to the Gramineaefamily. Incidentally, the “plant cell” of the present invention includesvarious forms of plant cells, such as suspension culture cells,protoplasts, a section from the leaf, callus, and so on.

For example, methods, like the callus differentiation method (Kyozuka,J. and Shimamoto, K. (1991) Plant Tissue Culture Manual. Kluwer AcademicPublishers, pp B1:1-16; Toki, S. (1997) Plant Molecular Biology Reporter15:16-21), the differentiation method utilizing protoplasts (Shimamoto,K. et al. (1989) Nature 338:274-276; Kyozuka, J. et al. (1987)Mol.Gen.Genet. 206:408-413), and such in response to the kind of plantused, can be utilized to regenerate transgenic plants from transgenicplant cells to which vectors are introduced.

Transgenic plants produced in this way show high Na⁺/H⁺ antiporteractivity as compared to wild-type plants, and are supposed to acquiresalt tolerance thereby. Moreover, once a transformed plant transfectedwith the DNA of the present invention is obtained, it is possible togain descendants from that plant body by syngenesis or agamogenesis.Alternatively, plants can be mass-produced from breeding materials (forexample, seeds, fruits, ears, tubers, tubercles, stubs, callus,protoplast, etc.) obtained from the plant, as well as descendants orclones thereof. Plant cells transferred with the DNA of the presentinvention, plant bodies including these cells, descendants and clones ofthe plant, as well as breeding materials obtained from the plant, itsdescendant and clones, are included in the present invention.

Such high Na⁺/H⁺ antiporter activity as compared to wild-type plants canbe achieved either by high expression of Na⁺/H⁺ antiporter (change inquantity) or by expression of Na⁺/H⁺ antiporter with higher activity(change in quality), or may be a result of both.

Further, the present invention provides antibodies binding to theproteins of the present invention described above. Both polyclonalantibodies and monoclonal antibodies are included in the presentinvention. Preparation of the antibody can be conducted according tomethods known to those skilled in the art, for example, using methods ofHarlow et al. (Harlow, E. and Lane, D. (1988) Antibodies: A laboratorymanual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).Polyclonal antibodies can be obtained by injecting fusion proteins,synthesized in E.coli or synthesized peptides, into a rabbit asantigens, obtaining rabbit antiserum, and purifying antibodies therefromby affinity chromatography. Monoclonal antibodies can be obtained byinjecting antigens to mouse or rats, cloning and preparing hybridomas,and subjecting thus obtained antibody to affinity chromatography.

Furthermore, the present invention provides nucleic acid molecules thathybridize with the DNA encoding the protein of the present invention,and which have a chain length of at least 15 nucleotides. Such nucleicacid molecules can be used, for example, as probes to detect or isolatethe DNA encoding the protein of the present invention, as well asprimers to enhance such DNA. Such nucleic acid molecules preferablyhybridize specifically to the DNA encoding the protein of the presentinvention. The term “hybridizes specifically” as used herein means thatit hybridizes to the DNA encoding the protein of the present inventionbut it does not hybridize to DNAs encoding other proteins under a normalhybridization condition, preferably under a stringent condition forhybridization.

In addition, such nucleic acid molecules can be used as antisenseoligonucleotides, ribozymes, and so on, that suppress expression of theprotein of the present invention. Derivatives and modified forms of theantisense oligonucleotides can be used in the same manner as theantisense oligonucleotide itself. The antisense oligonucleotide does nothave to be completely complementary to the nucleotides constituting thegiven region of the DNA or mRNA, and may include 1 or more nucleotidemismatches, provided it can suppress expression of the protein. Anantisense oligonucleotide and a ribozyme that suppresses expression of aprotein of the present invention can be a very useful tool for thefunction analysis of the protein of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the base sequence and the predicted amino acid sequence ofthe rice Na⁺/H⁺ antiporter (OsNHX1) cDNA. The amino acid sequence isexpressed in one letter notation.

FIG. 2 shows the hydrophobicity plot of the amino acids of the OsNHX1protein. The abscissa indicates the amino acid residue, and the ordinateindicates the degree of hydrophobicity. Predicted transmembrane regionsare shown as boxed numbers.

FIG. 3 shows the amino acid sequence comparison between OsNHX1 and otherNa⁺/H⁺ antiporters. Transmembrane regions (M3 to M6) are indicated abovethe sequence. Regarding the symbols under amino acids, “*” representsthat all amino acids are conserved; “:” and “.” represent that aminoacid are similar. “:” indicates much more similarity than thoseindicated by “.”. The box with A represents the binding site of thespecific inhibitor, amiloride, and the boxes with B represent sites withhigh identities to the mammalian Na⁺/H⁺ antiporter.

FIG. 4 shows the result of phylogeny analysis of Na⁺/H⁺ antiporter usingClustalX (Thompson, J. D. et al. (1994) Nucleic Acids Research,22:4673-4680) (Neighbor Joining (NJ) method).

FIG. 5 shows photographs indicating effects of hygromycin and NaCl onbudding yeast NHX1 mutant strain (Δnhx1) expressing OsNHX1 gene. WT wasK601 (wild-type) containing pKT10 (+HIS3), nhx1 was R100 (Δnhx1)containing pKT10 (+HIS3), and nhx1 GAP:OsNHX1 was R100 containing pKT10(+HIS3)+OsNHX1. Five-fold serial dilutions (starting at no dilution) ofthe indicated strains were grown at 28° C. for 3 days on YPD agar mediumsupplemented with or without hygromycin or for 8 days on APG agar medium(pH 4.0) supplemented with or without NaCl.

FIG. 6 shows a photograph of electrophoresis indicating results ofWestern analyses for OsNHX1 protein in a microsomal fraction of buddingyeast NHX1 mutant strain (Δnhx1) and a membrane fraction of rice. TM,TP, and PM indicate total membrane, tonoplast, and plasma membrane,respectively. The microsomal fraction of budding yeast was prepared fromR100 and R100 into which pKT10 (+HIS3)+OsNHX1 had been introduced. Themembrane fraction of rice was prepared from roots and shoots of youngrice plants hydroponically cultivated for seven days. The proteinsprepared (10 μg) were electrophoresed on a 10% SDS gel and subjected toWestern analyses with OsNHX1 protein-specific antibody and vacuolarH⁺-pyrophosphatase-specific (V-PPase-specific) antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

Any patents, patent applications, and publications cited herein areincorporated by reference.

The present invention will be specifically explained with reference tothe following examples. However, it should be noted that the presentinvention is not limited by these examples.

EXAMPLE 1

Cloning of the Rice Na⁺/H⁺ Antiporter Gene

A sequence having identity to Na⁺/H⁺ antiporter (NHX1) obtained frombudding yeast was analyzed from the database for higher plants ofGeneBank. A cDNA clone from the cDNA library of rice panicle wasidentified. The amino acid sequence predicted from the clone had 37%identity with NHX1. It was presumed that the obtained cDNA clone did nothave the full-length base sequence. Therefore, using the cDNA clone as aprobe and using the cDNA library constructed from mRNA prepared from theroot of rice (Oryza sativa L. cv Nipponbare) seedling as the template,selection of a cDNA clone having the full-length insertion wasperformed.

Rice seeds were imbibed overnight, and placed on cotton mesh suspendedover a nutrient solution (0.5 mM NH₄H₂PO₄, 1 mM KNO₃, 0.5 mM MgSO₄, 12.5μM Fe-EDTA, 1 mM CaCl₂, micronutrients) Cultivation was performed 7 dayswith a cultivation condition: day(brightness 40 μmol m⁻² s⁻¹) for 14hours at 30° C., night for 10 hours at 25° C., humidity at 75%.

Poly (A⁺) RNA from the root of rice seedling was prepared and sizefractionated by 5 to 25% sucrose density-gradient by centrifugation.Then, a cDNA library was constructed from the fractions containingrelatively large poly (A⁺) RNAs (Tanaka, Y. et al. (1989) Plant Physiol.90:1403-1407). Double stranded cDNA was synthesized from size fractionedpoly (A⁺) RNA by the method of Gubler and Hoffman (Gubler, U. andHoffman, B. J. (1983) Gene 25:263-269), using oligo dT as the primer.The sample was then size fractioned by high performance liquidchromatography (Tosoh, Tokyo, model CCPD,) using Asahipack GS710 column(Asahi Chemical Industry Co. Ltd., Tokyo; 2.5×50 cm). cDNAs larger than2 kb were inserted to the EcoRI site of λgt11.

Plaque hybridization was conducted using constructed λ phages havingcDNA libraries, and cDNA clones that show identity with the NHX1 asprobes. Selecting a vector with the longest cDNA insert from the plaquesthat showed signal, cloning was performed by inserting the cutout cDNAinto a pBluescript (KS+)vector (Stratagene). Confirmation that theobtained cDNA clone is a full-length cDNA was made by the signal sizefrom the Northern hybridization using RNAs extracted from the rice plantbody and the obtained clone as the probe. All base sequence of the cDNA,to which the whole isolated gene (referred to as OsNHX1) is inserted,was determined (FIG. 1).

EXAMPLE 2

Base Sequence and Amino Acid Sequence Analysis of OsNHX1 Gene

The full-length sequence was 2330 base pairs, the 5′ untranslated regionwas 296 base pairs, the translated region was 1608 base pairs and the 3′untranslated region was 426 base pairs. The protein encoded by OsNHX1was predicted to be 535 amino acids long, and the molecular weight wascalculated to be 59,070 daltons. 59% of the predicted amino acidssequence was hydrophobic, 22% was neutral amino acids, and 19% washydrophilic amino acids. Thus, the protein seemed to be highlyhydrophobic. The result of hydrophobicity analysis, by the method ofKyte and Doolittle (Kyte, J. and Doolittle, R. F. (1982) J.Mol.Biol.157:105-132), is shown in FIG. 2. Twelve transmembrane regions weredetected by the method of TMpred program (Hofmann, K. and Stoffel, W.(1993) Biol.Chem. Hoppe-Seyler 374:166).

Significant identity was detected for the amino acid sequence predictedfrom OsNHX1 with the amino acid sequence of NHX1 and mammalian Na⁺/H⁺antiporter (NHE) (Table 1; NHX1 in the table represents that derivedfrom yeast [S. cerevisiae], NHE6 from human, and NHE1 to 4 from rat. Thevalues on the table were calculated by the homology-analyzing program(Lipman, D. J. and Pearson, W. R. (1985) Science 227:1435-1441) ofGENETYX (ver.10) software (Software Development Company)). Especiallyhigh identity was observed in the transmembrane regions, which weresuspected to be involved in ion transport (FIG. 3). ⁸³LFFIYLLPPI⁹², apart of the amino acid sequence of OsNHX1 (residues 83-92 of SEQ IDNO.2), is very well conserved in NHX1 and NHE and is expected to be thebinding site of amiloride, an inhibiter of the eucaryotic Na⁺/H⁺antiporter (Counillon, L. et al. (1993) Proc.Natl.Acad.Sci.USA90:4508-4512) (FIG. 3A). In addition, the 6th and 7th transmembraneregions are well preserved in eucaryotic Na⁺/H⁺ antiporter and, thus, ispredicted to play an important role in the transport of Na⁺ and H⁺(Orlowski, J. and Grinstein, S. (1997) J.Biol.Chem. 272:22373-22376).The 5th and 6th transmembrane regions in the amino acid sequence ofOsNHX1 showed high identity to these regions (FIG. 3B). The aboveresults indicate that the protein encoded by OsNHX1 has the activity ofNa⁺/H⁺ antiporter.

TABLE 1 Amino acid sequence identity of OsNHX1 to otherNa⁺/H⁺antiporters (%) OsNHX1 NHX1 NHE6 NHE1 NHE2 NHE3 NHE4 OsNHX1 10029.5 33.0 30.1 29.4 26.7 27.7 NHX1 100 36.1 28.6 29.1 29.3 32.0 NHE6 10031.9 29.1 31.8 28.6 NHE1 100 48.9 37.1 45.5 NHE2 100 44.7 66.0 NHE3 10044.6 NHE4 100

Dendrogram of various Na⁺/H⁺ antiporters reported to date, namelymammalian NHE, budding yeast (S.cerevisiae) NHX1 and NHA1, Sod2 (whichis expected to be expressed on the plasma membrane of fission yeast,S.pombe), yeast (Zygosaccharomyces rouxii) ZSod3, E. coli NhaA and NhaB,as well as OsNHX1 (noted as “OsNHX1” in the figures) made according toNJ method, revealed that three of them, that is NHX1, NHE6 and OsNHX1,form a cluster (FIG. 4). It has been reported that NHX1 protein isexpressed in the late endosome (Nass, R. and Rao, R. (1998) J.Biol.Chem.273:21054-21060), and it was indicated that NHE6 protein is alsoexpressed in the cell (Numata, M., Petrecca, K., Lake, N. and Orlowski,J., J.Biol.Chem. 273:6951-6959). Therefore, it is expected that OsNHX1protein is expressed in the intracellular organs, like the vacuole andso on, and plays an important role in Na⁺ transport in these organs.

EXAMPLE 3

Production of Transformed Rice Expressing Rice Na⁺/H⁺ Antiporter Gene

OsNHX1 inserted in the BamHI site of pBluescript KS+ (STRATAGENE) wasexcised with KpnI and NotI. Then, OsNHX1 was inserted downstream of thecauliflower mosaic virus 35 S promoter of pMSH1 (for high expression)and pMSH2 (for repressed expression), both of which are derived fromTi-plasmid and are transferred with kanamycin resistance gene andhygromycine resistance gene (pMSH1: Kawasaki, T. et al. (1999)Proceedings of the National Academy of Sciences of the U.S.A.96:10922-10926; pMSH2: the multi cloning site has the opposite directioncompared to pMSH1). Using the constructed vector, the rice callus wastransformed with Agrobacterium tumefaciens. The callus was induced fromthe seed, and screened after the infection with Agrobacterium wascomplete using hygromycine. The screened callus was differentiated toobtain the transformant plant. Transformation and differentiation werebasically performed according to the method of Toki (Toki, S. (1997)Plant Molecular Biology 15,16-21).

EXAMPLE 4

Functional Complementation Experiments in Yeast

Experiments for functional complementation by OsNHX1 gene were performedusing budding yeast vacuolar Na⁺/H⁺ antiporter gene NHX1 mutant strain(Δnhx1, R100) (Nass, R. et al., (1997) The Journal of BiologicalChemistry 272, 26145-26152). Budding yeast was cultured in YPD medium,SD medium, or, in the case of NaCl treatment, APG medium. OsNHX1 genewas inserted downstream of the GAP promoter of pKT10 vector, into whichHIS3 gene was inserted. The resulting vector was introduced into buddingyeast by lithium method. NaCl and hygromycin-sensitivity of Δnhx1 wasrecovered by overexpressing OsNHX1 gene (FIG. 5). Thus, it was confirmedthat OsNHX1 gene encoded a protein having vacuolar Na⁺/H⁺ antiporterfunction.

EXAMPLE 5

Localization of OsNHX1 Protein

A peptide synthesized on the basis of the carboxyl-terminal 16 aminoacids of the amino acid sequence deduced from OsNHX1 gene was injectedinto a rabbit as an antigen. Antisera obtained were purified by affinitychromatography to prepare OsNHX1 protein-specific polyclonal antibody.Antibody preparation was requested from Sawady Technology Co., LTD. ByWestern analyses using the OsNHX1 protein-specific antibody obtained, itwas confirmed that OsNHX1 protein was largely localized in the tonoplastfraction (FIG. 6). Thus, it was confirmed that OsNHX1 protein was avacuolar Na⁺/H⁺ antiporter.

Industrial Applicability

According to the present invention, it is expected that isolated Na⁺/H⁺antiporter gene can render salt tolerance to the plant by expressing itin the plant. Therefore, it may conduce, for example, an increase in theharvest of crops, due to improvements in salt tolerance, by transferinto useful crops such as rice, which will make them resistant to harmby salt in dry land and such.

1. An isolated DNA selected from the group consisting of: (a) a DNAencoding a protein comprising the amino acid sequence described in SEQID NO.: 2; and (b) a DNA comprising the coding region of the nucleotidesequence described in SEQ ID NO.:
 1. 2. An isolated DNA encoding aprotein having an Na+/H+ antiporter activity obtained frommonocotyledonous plant selected from the group consisting of: (a) a DNAencoding a protein comprising the amino acid sequence described in SEQID NO.: 2, wherein the number of amino acids that are substituted,deleted, inserted and/or added is 20 or less; and (b) a DNA specificallyhybridizing under highly stringent conditions to the DNA consisting ofthe nucleotide sequence described in SEQ ID NO.: 1, wherein highlystingent conditions comprise washing at 56° C. in a wash solutioncontaining 0.1×SSC and 0.1% SDS.
 3. The isolated DNA of claim 2, whereinthe monocotyledonous plant belongs to the Gramineae family.
 4. A vectorcomprising DNA selected from the group consisting of: (a) a DNA encodinga protein comprising the amino acid sequence described in SEQ ID NO.: 2;and (b) a DNA comprising the coding region of the nucleotide sequencedescribed in SEQ ID NO.:
 1. 5. A vector comprising a DNA encoding aprotein having an Na+/H+ antiporter activity obtained from amonocotyledonous plant selected from the group consisting of: (a) a DNAencoding a protein comprising the amino acid sequence described in SEQID NO.: 2, wherein the number of amino acids that are substituted,deleted, inserted and/or added is 20 or less; and (b) a DNA specificallyhybridizing under highly stringent conditions to the DNA comprising thesequence described in SEQ ID NO.:1, wherein highly stingent conditionscomprise washing at 56° C. in a wash solution containing 0.1×SSC and0.1% SDS.
 6. A transformant cell transformed with a DNA selected fromthe group consisting of: (a) a DNA encoding a protein comprising theamino acid sequence described in SEQ ID NO.: 2; and (b) a DNA comprisingthe coding region of the nucleotide sequence described in SEQ ID NO.: 1.7. The transformant cell of claim 6, wherein the cell is a plant cell.8. A transformant cell transformed with a DNA encoding a protein havingan Na+/H+ antiporter activity obtained from a monocotyledonous plantselected from the group comprising: (a) DNA encoding a proteinconsisting of the amino acid sequence described in SEQ ID NO.: 2,wherein the number of amino acids that are substituted, deleted,inserted and/or added is 20 or less; and (b) a DNA specificallyhybridizing under highly stringent conditions to the DNA comprising thenucleotide sequence described in SEQ ID NO.:1, wherein highly stingentconditions comprise washing at 56° C. in a wash solution containing0.1×SSC and 0.1% SDS.
 9. The transformant cell of claim 8, wherein thecell is a plant cell.
 10. A transformant plant comprising a transformantcell transformed with a DNA selected from the group consisting of: (a) aDNA encoding a protein comprising the amino acid sequence described inSEQ ID NO.: 2; and (b) a DNA comprising the coding region of thenucleotide sequence described in SEQ ID NO.:
 1. 11. The transformantplant of claim 10, wherein the plant is a monocotyledon.
 12. Thetransformant plant of claim 11, wherein the monocotyledon belongs to theGramineae family.
 13. The transformant plant of claim 12, wherein theplant is rice.
 14. A transformant plant that is the offspring or cloneof a transformant plant comprising a transformant cell transformed witha DNA selected from the group consisting of: (a) a DNA encoding aprotein comprising the amino acid sequence described in SEQ ID NO.: 2;and (b) a DNA comprising the coding region of the nucleotide sequencedescribed in SEQ ID NO.: 1; wherein said offspring or clone carries saidDNA.
 15. A transformant plant comprising a transformant cell transformedwith a DNA encoding a protein having an Na+/H+ antiporter activityobtained from a monocotyledonous plant selected from the groupconsisting of: (a) a DNA encoding a protein consisting of the amino acidsequence described in SEQ ID NO.: 2, wherein the number of amino acidsare substituted, deleted, inserted and/or added is 20 or less; and (b) aDNA specifically hybridizing under highly stringent conditions to theDNA comprising the nucleotide sequence described in SEQ ID NO.:1,wherein highly stingent conditions comprise washing at 56° C. in a washsolution containing 0.1×SSC and 0.1% SDS.
 16. The transformant plant ofclaim 15, wherein the plant is a monocotyledon.
 17. The transformantplant of claim 16, wherein the monocotyledon belongs to the Gramineaefamily.
 18. The transformant plant of claim 17, wherein the plant isrice.
 19. A transformant plant that is the offspring or clone of atransformant plant comprising a transformant cell transformed with a DNAencoding a protein having an Na+/H+ antiporter activity obtained from amonocotyledonous plant selected from the group consisting of: (a) a DNAencoding a protein comprising the amino acid sequence described in SEQID NO.: 2, wherein the number of amino acids are substituted, deleted,inserted and/or added is 20 or less; and (b) a DNA specificallyhybridizing under highly stringent conditions to the DNA comprising thenucleotide sequence described in SEQ ID NO.:1, wherein highly stingentconditions comprise washing at 56° C. in a wash solution containing0.1×SSC and 0.1% SDS; wherein said offspring or clone carries said DNA.20. A material for the breeding of a transformant plant comprising atransformant cell transformed with a DNA selected from the groupconsisting of: (a) a DNA encoding a protein comprising the amino acidsequence described in SEQ ID NO.: 2; and (b) a DNA comprising the codingregion of the nucleotide sequence described in SEQ ID NO.:
 1. 21. Amaterial for the breeding of a transformant plant comprising atransformant cell transformed with a DNA encoding a protein having anNa+/H+ antiporter activity obtained from a monocotyledonous plantselected from the group consisting of: (a) a DNA encoding a proteincomprising of the amino acid sequence described in SEQ ID NO.: 2,wherein the number of amino acids that are substituted, deleted,inserted and/or added is 20 or less; and (b) a DNA specificallyhybridizing under highly stringent conditions to the DNA comprising ofthe nucleotide sequence described in SEQ ID NO.:1, wherein highlystingent conditions comprise washing at 56° C. in a wash solutioncontaining 0.1×SSC and 0.1% SDS.
 22. A isolated nucleic acid moleculehaving a chain length of at least 15 nucleotides that is identical to anat least 15-nucleotide fragment of the DNA described in SEQ ID NO.: 1.